Antibiotic complex producing bacterial culture

ABSTRACT

A novel anthracycline antibiotic complex designated herein as figaroic acid complex is produced by fermentation of Streptosporangium sp. strain C-31,751, A.T.C.C. 31129. Figaroic acid complex inhibits the growth of various microorganisms, e.g., Staphylococcus aureus, exhibits phage inducing properties and inhibits the growth of various tumors in rodents, e.g., Sarcoma 180, L-1210 lymphatic leukemia, B-16 melanoma, Walker 256 carcinosarcoma and P-388 lymphatic leukemia.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional application of co-pending application Ser. No.832,034 filed Sept. 9, 1977, now U.S. Pat. No. 4,112,071, which in turnis a continuation-in-part application of Ser. No. 758,248 filed January10, 1977 and now abandoned, which in turn is a continuation-in-partapplication of Ser. No. 590,994 filed June 27, 1975 and now abandoned.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

This invention relates to a new anthracycline antibiotic complex and toits production and recovery.

(2) Description of the prior art

A number of anthracycline glycosides have been described in theliterature. Among them, daunomycin and adriamycin are particularly beingwatched with keen interest in the field of cancer chemotherapy and havealready been applied clinically for human cancers.

Farmitalia's U.S. patent on adriamycin (B-106FI; 14-hydroxy-daunomycin;INN is Doxorubicin) is 3,590,028 claiming the product by structure anddisclosing its direct fermentation by S. peuceticus var. caesius.Farmitalia also issued U.S. Pat. No. 3,803,124 on chemical conversion ofdaunomycin to adriamycin; for direct fermentation of daunomycin (asantibiotic FI 1762) by S. peuceticus see U.K. 1,003,383.

Farmitalia's daunomycin (U.K. Pat. No. 1,003,383) may be the same asRhone-Poulenc's 13057 R.P. (formerly rubidomycin and now Daunoribicin(U.K. Pat. Nos. 985,598; 1,188,262; 1,241,750 and see U.S. Pat. No.3,616,242) and is "probably" identical with Ciba's danubomycin (U.S.Pat. No. 3,092,550; U.K. 901,830). See also U.S. Pat. No. 3,686,163 ondihydrodaunomycin.

Cinerubin A and cinerubin B are disclosed in U.K. Pat. No. 846,130 andsee also U.S. Pat. No. 3,864,480 and Keller-Schierlein et al.,Antimicrobial Agents and Chemotherapy, page 68 (1970) and ChemicalAbstracts, 54, 1466i (1960).

The anthracycline glycoside carminomycin described in J. Antibiotics,April 1974, pages 254-259, has been reported to be active againstseveral animal tumor systems.

For further illustrative and summary disclosures of anthracyclineantibiotics see Index of Antibiotics from Actinomycetes, Hamao Umezawa,Editor-in chief, University Park Press, State College, Pennsylvania,U.S.A. (1967) as follows:

    ______________________________________                                        Antibiotic           Page Number                                              ______________________________________                                        Aklavin              111                                                      Cinerubin A          220                                                      Cinerubin B          221                                                      Danubomycin          242                                                      Daunomycin           243                                                      Pyrromycin           542                                                      Rhodomycin A,B       561                                                      Rubidomycin          574                                                      ______________________________________                                    

The textbook Antibiotics, Volume 1, Mechanism of Action, edited by DavidGottlieb and Paul D. Shaw, Springer--Verlag New York, Inc. N.Y., N.Y.(1967) at pages 190-210 contains a review by A. DiMarco entitledDaunomycin and Related Antibiotics.

Information Bulletin, No. 10, International Center of Information ofAntibiotics, in collaboration with WHO, Dec., 1972, Belgium, reviewsanthracyclines and their derivatives.

SUMMARY OF THE INVENTION

There is provided by the present invention a new anthracyclineantibiotic complex designated herein as figaroic acid complex, saidcomplex being prepared by cultivating a new strain of Streptosporangiumdesignated Streptosporangium sp. strain C-31,751, A.T.C.C. No. 31129, inan aqueous nutrient medium containing assimilable sources of nitrogenand carbon under submerged aerobic conditions until a substantial amountof figaroic acid complex is produced by said organism in said culturemedium and optionally recovering the figaroic acid complex from theculture medium. The invention embraces the unresolved mixture ofanthracycline antibiotics designated as figaroic acid complex in dilutesolution, as crude concentrates or in solid form.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the infra-red absorption spectrum of figaroic acid complex(KBr pellet).

FIG. 2 shows the ultraviolet absorption spectra of figaroic acid complexin 0.1N HC1 in methanol (solid line) and in 0.1N NaOH in methanol(dotted line).

DETAILED DESCRIPTION

This invention relates to a novel anthracycline antibiotic complexdesignated herein as figaroic acid complex and to its preparation byfermentation of a new strain of Streptosporangium designatedStreptosporangium sp. strain C-31,751. The above organism was obtainedfrom a soil sample taken from Seelyville, Indiana. A culture of theorganism has been deposited without restrictions in the American TypeCulture Collection, Washington, D.C., and added to its permanentcollection of microorganisms as A.T.C.C. 31129.

Figaroic acid complex inhibits growth of various Gram-positive bacteria,for example, Staphylococcus aureus and Mycobacterium tuberculosis, andvarious protozoa and yeasts, for example, Candida albicans, Histoplasmacapsulatum, Trichomonas vaginalis and Trichomonas faetus. The substanceexhibits phage inducing properties and inhibits growth of variouslymphatic and solid tumor systems in rodents including Sarcoma 180,L-1210 lymphatic leukemia, Walker 256 carcinosarcoma, P-388 lymphaticleukemia and B-16 melanoma. The figaroic acid complex may be used aloneor in combination with other antibacterial agents to prevent the growthof, or reduce the number of, the sensitive Gram-positive bacteria,yeasts and protozoa mentioned above. It is useful in wash solutions forsanitation purposes, e.g., for washing hands and disinfecting variouslaboratory, dental and medical equipment or other contaminated materialsand as a bacteriostatic rinse for laundered clothes. It is also usefulin treating the above-mentioned tumor systems in mice and rats.

THE MICROORGANISM

The strain C-31,751 has the following morphological characteristics:Scant aerial mycelium are formed. During the early growth phase, a sporechain which is short, compact and irregularly coiled appears at the tipof the sporophore. The coiled spore chain develops into a realsporangium which is spherical in shape and 4-12μ in diameter. Most ofthe sporangiophores measure 5-10μ in length. Looped or short flexuousspore chains are occasionally co-produced with the sporangia. Thesubstrate mycelium is branched, often curved and probably non-septated.The sporangiospore is non-motile, spherical to oval in shape and0.7-0.9μ in size. The spore-surface structure has not yet beendetermined.

Table I reports the cultural properties obtained on different media, theobservations being after 1-2 months culture at 28° C. The organism formsaerial mycelium slowly on sucrose-nitrate agar, inorganic salts-starchagar, yeast extract-malt extract agar and oat meal agar. Mass color ofthe aerial mycelium is whitish pink to pink. Aerial mycelium was notformed on asparagine agars, tyrosine agar, nutrient agar andpeptone-yeast extract-iron agar.

The sporangium is formed on inorganic salts-starch agar, yeastextract-malt extract agar and oat meal agar. Numerous sporangia wereseen on the latter two media after incubation for seven weeks at 28° C.The mass of substrate mycelium shows granular shape on macro- andmicroscopic observation. The principal color of the substrate myceliumis reddish orange and reddish purple on glucose-asparagine agar andyeast extract-malt extract agar, respectively. Light yellowishdiffusible pigment is produced in glucose-asparagine agar and yeastextract-malt extract agar. A trace amount or no melanoid pigment isproduced in tyrosine agar and peptone-yeast extract-iron agar.

                                      TABLE 1                                     __________________________________________________________________________    Cultural Characteristics Of Strain C-31751*                                                                            Diffusible                                          Growth  Reverse color**                                                                        Aerial mycelium                                                                        Pigment                              __________________________________________________________________________    Sucrose-nitrate agar                                                                         Scant, fine                                                                           Pink     Very scant,                                                                            None                                                granular         pinkish white                                 Glucose-asparagine agar                                                                      Moderate,                                                                             Strong reddish                                                                         None     None or light                                       granular                                                                              orange            reddish yellow                       Glycerol-asparagine agar                                                                     Scant, fine                                                                           Strong orange                                                                          None     None                                                granular                                                       Inorganic salts-starch agar                                                                  Moderate,                                                                             Vivid yellowish                                                                        Scant, whitish                                                                         None                                                granular                                                                              orange                                                 Tyrosine agar  Scant, fine                                                                           Light, yellowish                                                                       None     None                                                granular                                                                              brown                                                  Nutrient agar  Moderate,                                                                             Dull reddish                                                                           None     None                                                granular                                                                              purple to dark                                                                wine                                                   Yeast extract-malt extract agar                                                              Good, crenate                                                                         Dark rose to deep                                                                      Scant, purplish                                                                        Light reddish                                               reddish purple                                                                         pink     yellow                               Oat meal agar  Moderate,                                                                             Purplish pink to                                                                       Scant, whitish                                                                         Purplish pink                                       granular                                                                              bright purple                                                                          to pale pink                                  Peptone-yeast extract-iron agar                                                              Moderate,                                                                             Dark violet                                                                            None     Gold                                 __________________________________________________________________________     *Observation after 1-2 month' culture at 28° C.                        **The pigment is soluble in methanol and has a pHindicator-like property:     yellowish orange at acidic and violet at alkaline pH.                    

The physiological characteristics and carbohydrate utilization of strainC-31,751 are shown in Tables 2 and 3, respectively. The organism reducesnitrate to nitrite in a natural organic medium but not in an inorganicmedium. Like most Micromonospora species, it is considerably sensitiveto sodium chloride. It is a mesophilic organism. Certain carbohydratessuch as sucrose, raffinose, soluble starch and D-mannitol are utilizedafter a long lag time.

                                      TABLE 2                                     __________________________________________________________________________    Physiological Characteristics Of C-31751                                      Tests        Responses     Materials And Methods                              __________________________________________________________________________    Nitrate reduction in                                                                       Negative      Czapek's sucrose-nitrate broth                     inorganic medium                                                              Nitrate reduction in                                                                       Strongly positive                                                                           The organic medium, recommended                    organic medium             by Leudemann.sup.1                                 Gelatin liquefaction                                                                       Completely liquified                                                                        Basal medium: Yeast extract                                                   0.4%, malt extract 1.0 and                                                    glucose 0.4%                                       Starch hydrolysis                                                                          Weakly positive                                                                             Hayward's starch agar                              Skim milk agar                                                                             Poor growth, weak                                                                           Leudemann's medium.sup.1                                        hydrolysis                                                       10% skim milk solution                                                                     Strong coagulation and                                                        slow peptonization                                               Melanin formation                                                                          Nor produced  Tyrosine agar and peptone-                                                    yeast extract-iron agar                            Effect of NaCl in organic                                                                  Moderate growth at 0 and                                                                    Basal medium: Leudemann's                          medium       0.5% NaCl. Restricted at                                                                    yeast: extract-starch                                           1 and 1.5%. No growth at                                                                    agar.sup.1                                                      2.5%.                                                            Growth-temperature                                                                         Maximum growth at 37° C.                                                             Yeast extract-malt extract                                      Medial growth at 28° C. and                                                          agar: ISP No. 2 medium                                          43° C. No growth at 15° C.                                      and 48° C.                                                __________________________________________________________________________     .sup.1 Leudemann, G. H.:Micromonospora purpureochromogenes (Waksman and       Curtis 1916) comb. nov. (Subjective Synonym:Micromonospora fusca Jensen       1932). Intl, J. Syst. Bacteriol. 21: 240247, 1971.                       

                  TABLE 3                                                         ______________________________________                                        Carbohydrate-utilization Of Strain C31751*                                             I**   II                 I     II                                    ______________________________________                                        D(-)-Arabinose                                                                            ±    -     D(-)-Melibiose                                                                          ++.sup.A                                                                             +.sup.A                            L(+)-Arabinose                                                                           ++      ++     Trehalose ++    ++                                  D-Xylose    ++.sup.A                                                                             ++     Raffinose +.sup.A                                                                             -                                   D-Ribose   ++      ++     D(-)-Mele-                                                                              -     -                                                             zitose                                              L-Rhamnose ++      ++     Soluble starch                                                                          +     ±                                D-Glucose  ++      ++     Cellulose +     ±                                D(+)-Galactose                                                                            ++.sup.A                                                                             ++     Glycerol  ++    ++                                  D-Fructose  +      ++     Inositol  ++.sup.A                                                                            ++                                  D-Mannose  ++      ++     D-Mannitol                                                                               +.sup.A                                                                            +.sup.A                             L(-)-Sorbose                                                                              +       -     D-Sorbitol                                                                              -     -                                   Sucrose      +.sup.A                                                                               +.sup.A                                                                            Dulcitol  -     -                                   Lactose     +       +     Salicin   ±  -                                   Cellobiose ++      ++     No sugar  -     -                                   ______________________________________                                         *Observation after 1-2 months' culture at 28° C.                       **Basal medium I: PridhamGottlieb medium plus 0.1% Difco yeast extract.       II: Leudemann's organic medium, composed of 0.5% yeast extract, 0.1%          CaCO.sub.3, and 1.5% agar in distilled water.                                 .sup.A Aerial mycelium poorly formed. No aerial mycelium on the other         sugar media.                                                             

Strain C-31,751 contains meso-diaminopimelic acid (meso-DAP) as acharacteristic amino acid component in the cell wall. Diagnosticcarbohydrate was not present .

Summarizing the above characteristics, strain C-31,751 forms whitishpink (shell pink) aerial mycelium and spherical sporangium. Thesporangiospore is not motile. The sporangiophore is short, usually lessthan 10μ in length. The mass color of the substrate mycelium is orangeto violet. Distinct diffusible pigment (including melanin) is notproduced. Almost all of the usual carbohydrates are utilized for thegrowth. The cell wall of the strain contained meso-DAP but no diagnosticsugar component.

These major characteristics indicate that strain C-31,751 is a speciesof the genus Streptosporangium. According to the taxonomicclassification of Streptosporangium species by H. Nonomura and Y. Ohara(J. Ferment. Technol. 47 (11): 701-709, 1969 and 52 (2): 71-77 (1974),sixteen species are described. Among them, eight species have pinkishaerial mycelium and short sporangiophore; they are Streptosporangiumrubrum Potekhina 1965, S. longisporum Schaffer 1969, S. roseum Couch1955, S. amethystogenes Monomura et Ohara 1960, S. amethystogenes var.nonreducens Prauser et Eckerdt 1967, S. vulgare Nonomura et Ohara 1960,S. pseudovulgare Nonomura et Ohara 1969 and S. nondiastaticum Nonomuraet Ohara 1969. Subsequently, S. violaceochromogenes MK-49 was added tothe same species-group (Japanese Patent 49-42896 of April 4, 1974).

Strain C-31,751 differs from Streptosporangium amethystogenes, S. roseumand S. vulgare in its positive growth at 42° C.; from S. longisporum inits glubose spore; from S. nondiastaticum in its positive utilization ofrhamnose, inositol and starch and from S. pseudovulgare in its positiveutilization of rhamnose and inositol and its orange or reddish purplesubstrate mycelium. S. violaceochromogenes is differentiated from StrainC-31,751 in its colorless or gold substrate mycelium and its negative ordoubtful utilization of inositol and rhamnose. Strain C-31,751 sharesseveral characteristics in common with Streptosporangium rubrumdescribed by L. L. Potekhina in Mokrobiologiya, 34, 292 (1965) such asthe aerial mass color, color of substrate mycelium, soluble pigment andspore-shape. However, the descriptions on S. rubrum presently availableare not sufficient to make a definite conclusion about the identity ofthe two organisms, and strain C-31,751 will therefore be considered anundetermined species of Streptosporangium until further data isavailable.

It is to be understood that for the production of figaroic acidantibiotic complex the present invention, though described in detailwith reference to the strain of Streptosporangium sp. strain C-31,751,A.T.C.C. 31129, is not limited to this microorganism or tomicroorganisms fully described by the cultural characteristics disclosedherein. It is intended that this invention also include other figaroicacid-producing strains or mutants of the said microorganisms which canbe produced by methods well known in the art, for example, by subjectingthe novel microorganism to x-ray or ultraviolet radiation, nitrogenmustard, phage exposure or the like.

PREPARATION OF THE COMPLEX

Figaroic acid complex is produced by cultivating a figaroicacid-producing strain of Streptosporangium having the characteristics ofA.T.C.C. 31129 or a mutant thereof under submerged aerobic conditions inan aqueous nutrient medium. The organism is grown in a nutrient mediumcontaining an assimilable carbon source, for example an assimilablecarbohydrate. Examples of preferred carbon sources include lactose,glycerol, sucrose, corn starch, glucose, mannose and fructose. Whenstarch is used as the carbon source in the nutrient medium, amylase maybe added to the broth before harvest to reduce any emulsion problemswhich may occur. The nutrient medium should also contain an assimilablenitrogen source such as, for example, fish meal, peptone, soybean flour,peanut meal, cotton seed meal and corn steep liquor. Nutrient inorganicsalts may also be incorporated in the medium, and such salts maycomprise any of the usual salts capable of providing sodium, potassium,ammonium, calcium, phosphate, sulfate, chloride, bromide, nitrate,carbonate or like ions.

Production of the figaroic acid complex can be effected at anytemperature conducive to satisfactory growth of the organism, i.e., roomtemperature up to about 43° C., and is conveniently carried out at atemperature of around 27° C. Ordinarily optimum production is obtainedafter incubation periods of about 170-210 hours. The medium normally isslightly alkaline, but the exact pH may vary according to the particularmedia used. The fermentation may be carried out in Erlenmeyer flasks andin laboratory or industrial fermenters of various capacities. When tankfermentation is to be carried out, it is desirable to produce avegetative inoculum in a nutrient broth by inoculating the broth culturewith a slant or soil culture or a lyophilized culture of the organism.After obtaining an active inoculum in this manner, it is transferredaseptically to the fermentation tank medium for large scale productionof the antibiotic complex. The medium in which the vegetative inoculumis produced can be the same, as, or different from, that utilized in thetank for the production of the new complex, as long as it is such that agood growth of the microorganism is obtained.

When the fermentation is complete, the antibiotic complex is extractedfrom the whole broth with a water-immiscible organic solvent, theorganic extract is concentrated, and the solid complex is precipitatedby dilution of the concentrated extract with a suitable antisolvent. Anywater-immiscible organic solvent having a polarity ranging from that ofmethylene chloride to that of n-butanol and having a pH in the range offrom about 3.5 to 8.5 may be used in the extraction step. Examples ofsuch water-immiscible solvents include C₄ -C₈ alcohols (n-butanol,sec-butanol, amyl alcohol, hexyl alcohol), higher ketones such as methylisobutyl ketone, chlorinated hydrocarbons such as methylene chloride andesters such as ethyl acetate. Solvents in the intermediate polarityrange of that indicated above such as ketones (e.g. methyl isobutylketone) and esters (e.g. ethyl acetate) are preferred since they arefound to be more selective than alcohols but polar enough to give gooddistribution characteristics. The most preferred water-immisciblesolvents are methyl isobutyl ketone and ethyl acetate. Extraction isconveniently done either under weakly acidic conditions, e.g. pH 4.0-5.0effected by addition of a mineral acid such as HCl or H₂ SO₄, or underweakly alkaline broth pH conditions, e.g. pH 8-8.5. Maximum yields havebeen obtained at pH 4.5-5.0 with methyl isobutyl ketone. Filter aid ispreferably added to the extraction mixture and the mixture thenfiltered. The organic phase is concentrated and diluted with anappropriate antisolvent (non-solvent for the complex) to precipitate outthe figaroic acid complex. Suitable antisolvents can be determined by asimple test and include such organic non-solvents for the complex asdiethyl ether, benzene and liquid aliphatic hydrocarbons such asn-hexane, n-heptane or Skellysolve B (isomeric hexanes). The preferredantisolvents are diethyl ether and n-hexane. If recovered under alkalineconditions, the purple figaroic acid complex may be converted to thered-orange free acid form by dissolving the complex in water andacidifying the solution to precipitate out the acid form of the complexwhich can then be recovered by filtration or extracted into organicsolvents.

Properties of Figaroic Acid Complex

The antibiotic complex designated herein as figaroic acid complex is anorange-red amorphous solid in the free acid state. It is insoluble inrelatively non-polar solvents such as diethyl ether, benzene andaliphatic hydrocarbons (e.g. n-hexane or n-heptane), mostly soluble inlower alcohols (i.e. C₁ -C₈ alcohols such as methanol, ethanol,n-butanol or -2-propanol), acetone, tetrahydrofuran and dioxane, andtotally soluble only in very polar solvents such as dimethylformamideand dimethylacetamide. Upon recovery under alkaline conditions, thecomplex is deep purple in color indicating conversion of the acid formto the anionic state.

Figaroic acid complex readily forms salts with organic and inorganicbases and pharmaceutically acceptable salts of the complex with suchbases are included within the scope of the present invention. Examplesof suitable pharmaceutically acceptable salts will be obvious to thoseskilled in the art and include those salts commonly employed in pharmacywith acidic drugs. The salts are readily obtained by reacting thecomplex with the appropriate organic or inorganic base. Illustrative ofthe salts which may be formed are metal salts such as zinc, aluminum,alkali metal (sodium or potassium) or alkaline earth metal (calcium,magnesium or barium), ammonium and organic amine salts such asethanolamine, ethylenediamine, diethanolamine, procaine ortriethanolamine.

The complex contains the elements carbon, hydrogen, oxygen and nitrogenin the following percentages by weight: carbon 53.82%, hydrogen 5.85%,nitrogen 1.63% and oxygen (by difference) 38.70%. It is soluble inaqueous NaHCO₃ and Ba(OH)₂ giving respectively red-violet and bluesolutions. It gives a deep red solution having red fluorescence withalcoholic magnesium acetate and a black solution (orange brown withviolet fluorescence on dilution) with alcoholic ferric chloride. Thecomplex gives a positive Tollens test but carbazole and ninhydrin testsare masked by the color of the pigment. It gives no color change withacid zinc dust, sodium bisulfite and hydrogen peroxide. There is aslight fading of the color from violet to red with alkaline sodiumbisulfite. Alkaline hydrogen peroxide gives no effect except in largeexcess whereupon the color fades from violet to pink.

Thin layer chromatograms were run on TLC plates precoated with SilicaGel 60F-254 (Brinkmann). Samples of the complex were made up atconcentrations of 2 mg./ml. in chloroform and 40 μl (80 μg) portionsthen applied to the plates. Chromatography was run in tanks having thewalls loosely lined with filter paper presaturated with the particularsolvent system used. Results (pigment zones were detected visually) wereas shown below for the three systems deemed definative. R_(f) values and(in parentheses) zone intensities, qualitatively estimated as strong(s), moderate (m) and weak (w), are indicated. Zones with an R_(f) of0.0 are at the point of application.

System A: chloroform: methanol (19:1) 9 zones noted: R_(f) 's=0.0 (s),0.39 (w), 0.214 (m), 0.243 (w), 0.349 (m), 0.470 (w), 0.612 (s),0.651(s) and 0.694 (m).

System B: chloroform: methanol: formic acid (90:10:1) 14 zones noted:R_(f) 's=0.0 (w), 0.043 (s), 0.073 (m), 0.149 (s), 0.169 (s), 0.212 (w),0.272 (m), 0.308 (m), 0.368 (w), 0.467 (m), 0.523 (m), 0.722 (s), 0.795(w) and 0.825 (s).

System C: chloroform: methanol: formic acid (80:20:1) 13 zones noted:R_(f) 's=0.0 (very weak), 0.200 (m), 0.233 (w), 0.279 (m), 0.311 (w),0.446 (m), 0.472 (s), 0.508 (m), 0.622 (w), 0.741 (m), 0.839 (s), 0.892(m) and 0.928 (s).

The infra-red and ultraviolet absorption spectra of figaroic acidcomplex indicate that the complex is a mixture of anthracyclinecomponents. The infra-red spectrum (KBr pellet) of FIG. 1 shows majorbands at 2.94 (broad), 3.4, 6.04-6.13, 6.18, 6.3, 6.95 (broad), 7.1(broad), 8.1, 9.3, 9.5 and 9.7μ. The ultraviolet absorption spectra ofthe complex under acidic and basic conditions are shown in FIG. 2.Adsorptivity in FIG. 2 is defined by the equation ##EQU1## where A isthe adsorbance, b is the cell width in cm. and c is the sampleconcentration in g./1. At a concentration of 50 μg./ml. in 0.1N HCl inmethanol, figaroic acid complex shows absorption peaks (solid line) at233, 253, 287-288 (shoulder), 467 (shoulder), 480 (shoulder), 490, 511(shoulder) and 524 (shoulder) m μ. In 0.1N NaOH in methanol the complexshows absorption peaks (dotted line) at 238, 266-268 (shoulder) and 553mμ.

One of the anthracycline components comprising the figaroic acid complexof the present invention has been determined to be carminomycin Ipreviously disclosed by M. G. Brazhnikova, et al. in J. Antibiotics, 27,254 (1974) as being prepared by fermentation of a species ofActinomadura, i.e. Actinomadura carminata (See also W. German OLS2,362,707). Carminomycin has been reported to show promising antitumoractivity in a variety of animal and human cancers [see, for example,Cancer Chemother. Rep., Part 1, 58, 255 (1974)].

Carminomycin I may be prepared according to the present invention bymild acid hydrolysis of figaroic acid complex. Thus, the figaroic acidcomplex may be extracted into an organic solvent, preferably methanol ora mixture of methanol and chloroform, and the organic extract thensubjected to mild acid hydrolysis to form carminomycin I which cansubsequently be recovered as by evaporation and purified, e.g. bychromatography over Sephadex LH-20. A detailed description of theconversion of figaroic acid complex to carminomycin I is provided inExamples 11-14 below.

Biological Activity Data

The in vitro minimum inhibitory concentrations (MIC) of figaroic acidcomplex were determined for a number of microorganisms using thestandard tube dilution procedure. The results shown in Table 4 indicatethat several gram-positive organisms, yeast and three protozoans weresensitive to the antibiotic. Gram-negative organisms were insensitive.

                  TABLE 4                                                         ______________________________________                                        Antimicrobial Spectrum of Figaroic Acid Complex                               Test organism            MIC,μg/ml                                         ______________________________________                                        Bacteria:                                                                     Staphylococcus aureus                                                                              A9537   1.6                                              Mycobacterium tuberculosis BCG                                                                     A9579   25                                               Escherichia coli      A15119 50                                               Pseudomonas aeruginosa                                                                             >˜50                                               Proteus mirabilis    A9900   >50                                              Salmonella enteritidis                                                                             A9531   >50                                              Yeasts:                                                                       Candida albicans     A9540   50                                               Trychophyton mentagrophytes                                                                        A9870   >50                                              Microsporum canis    A9872   >50                                              Protozoa:                                                                     Histoplasma capsulatum                                                                              A15056 6.3                                              Trichomonas vaginalis                                                                               A20074 1.25                                             Trichomonas faetus    A20075 0.31                                             ______________________________________                                    

Figaroic acid complex were tested for its ability to inducebacteriophage production in the lysogenic strain of Escherichia coli W1709. Significant induction was observed down to 0.8 μg/ml. Tubedilution protein tests to determine cytoxic effects on He La cells intissue culture gave a 50% end-point (ED₅₀) of 0.004 μg/ml (methoddescribed in Antimicrobial Agents and Chemotheropy; 1966: 613-618,1967).

The effect of figaroic acid complex on several rodent tumor systems werealso studied. Details of the methods used have been described in CancerResearch 22: 167-173, 1962 and Cancer Chemoth. Reports 3: 1-87(Part 3),1972. Treatment of mice having Sarcoma 180 implanted subcutaneously as asolid tumor with figaroic acid complex fermentation broth caused 37%inhibition of tumor diameter increase (estimated 75% inhibition in tumorweight increase). Treatment with the same broth also increased the lifespan of mice bearing L-1210 leukemia by 29% over control animals. Thefigaroic acid complex broth was found to be active against Walker 256carcinosarcoma (intramuscular), P-388 lymphatic leukemia and B-16melanoma in rodents. Solid figaroic acid complex was also tested andfound active on various tumor systems. Results on L-1210 leukemia andB-16 melanoma in mice are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                        Effects of figanoic acid complex on transplanted mouse tumors                        L-1210 leukemia   B 16 melanoma                                                 Aug. Wt  T/C          Aug. Wt                                                                              T/C                                              differ-  per-   Survi-                                                                              differ-                                                                              per- Survi-                             Dose     ence     cent   vor   ence   cent vors                               ug/Kg/day                                                                              (T-C,g)  MST    Day 5 (T/C,g)                                                                              MST  Day 5                              ______________________________________                                        128      -5.2     Tox    1/6   -3.4   Tox  6/6                                64                             0      Tox  6/6                                32       -2.8     150    6/6   -3.4    254.sup.(1)                                                                       6/6                                16                             -3.7   >400.sup.(2)                                                                       6/6                                8        -2.6     129    6/6   -0.9   206  6/6                                4                              +3.6   160  6/6                                2        -0.7     114    6/6   +0.9   126  6/6                                1                              -1.7   129  6/6                                Treatment: Once daily for 9 days, intraperitoneally                           Evaluation: T/C percent MST = median survival time in days:                   Treated MST/Control MST × 100.                                          Criteria: T/C.sup.= > 125 considered significant tumor inhibition             (prolongation of host survival)                                               ______________________________________                                         .sup.(1) 2/6 survivors at 60 days                                             .sup.(2) 4/6 survivors at 60 days                                        

The following examples serve to illustrate the invention withoutlimiting it. Skellysolve B is a commercially available petroleum solvent(Skelly Oil Co.) comprising isomeric hexanes and having a b.p. of60°-68° C. The main component of Skellysolve B is n-hexane. MIBK ismethyl isobutyl ketone.

EXAMPLE 1 Shake-flask fermentation

The organism Streptosporangium sp. strain C-31,751 is grown on an agarslant medium consisting of 2 g. D-glucose, 20 g. oatmeal, 2 g. soypeptone and 20 g. agar made up to one liter with distilled water. Afterat least 6 days growth at 27° C., spores are transferred to a 500 ml.Erlenmeyer flask containing 100 ml. of sterile medium consisting of 50g. corn starch, 10 g. soy flour, 10 g. peanut meal and 3 g. CaCO₃ madeup to one liter with distilled water. This vegetative culture isincubated at 27° C. on a rotary shaker (Gyrotory tier shaker, Model G53,New Brunswick Scientific Co., Inc.) set at 210 rev./min. describing acircle with a 5.1 cm. diameter. After 48 hours 4 ml. of culture istransferred to a 500 ml. Erlenmeyer flask containing 100 ml. of sterileproduction medium consisting of 50 g. sucrose, 20 g. soy flour, 20 g.peanut meal and 3 g. CaCO₃ made up to one liter with distilled water.The culture is incubated at 27° C. on a shaker set at 230 rev./min. for170 hours. At this time antibiotic activity consisting of the figaroicacid complex is found in the culture filtrate and mycelium.

EXAMPLE 2 Tank fermentation

A tank fermentor with 37.8 liters of sterile production medium (as inExample 1) is inoculated with 1.89 liters of vegetative culture preparedaccording to Example 1, agitated with an impeller speed of 375rev./min., aerated at a rate of 76.5 liters/min. and incubated at 27° C.After 190 hours the antibiotic complex is isolated.

EXAMPLE 3 Tank fermentation

A tank fermentor with 3030 liters of production medium (as in Example 1)is inoculated with 152 liters of vegetative culture (as prepared byExample 1), agitated with an impeller speed of 155 rev./min., aerated ata rate of 1420 liters/min. and incubated at 27° C. The figaroic acidcomplex is isolated after 210 hours.

EXAMPLE 4 Extraction of Broth and Mycelia at broth pH (slightlyalkaline) with n-butanol

Filtration of a broth from a shake flask fermentation using a total of 5liters of starting medium gave 2.5 liters filtrate, pH 8.5. Of this 1.5liters was extracted two times with 1 liter batches of n-butanol, thephases separated, and the combined organic phases concentrated. Dilutionof the concentrate with diethyl ether gave 1.4 g. of an amorphous purplesolid, active against L-1210 leukemia in mice at 2 mg./kg./day. Themycelial cake from broth filtration was stirred for 30 min. with enoughmethanol to obtain a fluid slurry and then filtered. The filtrate wasconcentrated until most of the alcohol had been removed and the aqueousresidue extracted as above to afford 6.35 g. of figaroic acid complex.The product, an amorphous purple solid, was found to be active vs.L-1210 leukemia in mice at a concentration of 8 mg./kg./day.

EXAMPLE 5 Extraction of whole broth at broth pH with n-butanol

Whole broth, 1.5 liters at pH 8.6, was stirred with about an equalvolume of n-butanol. The thick mass was filtered through a Celite(trademark of diatomaceous earth produced by Johns-Manville ProductsCo.) cake, the phases separated, and the organic phase concentrated to asmall volume. This was diluted with excess diethyl ether to precipitate621 mg. of figaroic acid complex. The amorphous purple solid is found tobe active against L-1210 leukemia in mice at a dosage of 0.2mg./kg./day. The solid does not melt but decomposes above 200° C.

EXAMPLE 6 Extraction of whole broth at acid pH with n-butanol

The general procedure of Example 5 was repeated except that pH of thewhole broth was adjusted to pH 4.0 with HCl and maintained there duringextraction. Four liters of whole broth yielded 1.7 g. of figaroic acidcomplex in the free acid state as an orange-red solid. The solid istoxic to mice at a concentration of 0.25 mg./kg./day and shows phageinducing properties down to a dilution of 1.5 μg./ml.

EXAMPLE 7 Extraction of whole broth (slightly alkaline) with n-butanolon a large scale

Whole broth (2788 liters at pH 8.6) was stirred with 1357 litersn-butanol. The organic phase was separated to give 783 liters of richextract which was concentrated to 13 liters. Addition of 80 liters"Skellysolve B" gave 728 g. of the crude purple figaroic acid complex.

EXAMPLE 8 Acid extraction of whole broth with methyl isobutyl ketone(MIBK)

Whole broth (10 liters) which had been stored frozen was thawed andstirred with 10 liters MIBK for 20 minutes after adjustment of the pH to4.5. Filter aid was stirred into the mixture and the latter was thenfiltered on a filter aid pad. The phases in the filtrate were separatedand the organic phase concentrated to a small volume. Dilution of thiswith excess "Skellysolve B" gave 4 g. of the orange-red figaroic acidcomplex free acid having phage inducing activity at a dilution of 6.2μg/ml.

EXAMPLE 9 Acid extraction of whole broth with methyl isobutyl ketone ona large scale (MIBK)

Whole broth (3095 liters) was adjusted from pH 8.35 to 3.35 at 10° C. byaddition of 49 liters 30% H₂ SO₄ with stirring and cooling. Two volumes(6412 liters) MIBK were stirred with the broth followed by addition ofexcess filter aid (diatomaceous earth). The mixture was filtered acrossa precoated vacuum filter using 2554 liters additional MIBK as rinse.The rich MIBK extract (7555 liters) was separated and concentrated to 10liters. During the concentration 1.285 kg. of wet solids precipitatedand were collected. Addition of 100 liters "Skellysolve B" to theconcentrate afforded an additional 365 g. of crude oily material. Thefirst crop of solids was dried to 858 g. and divided into three batches.Each of these was stirred in 4 liters acetone and insoluble matterfiltered off. The latter proved to be filter aid and, when combined anddried, weighed 367 g. The acetone solution was concentrated and dilutedwith excess diethyl ether to give 294.5 g. of crude figaroic acidcomplex. Evaporation of the ether gave 19.2 g. of inactive material. Theoily "Skellysolve B" precipitate was treated also with diethyl ether,but proved inactive.

EXAMPLE 10 Conversion of figaroic acid complex salt to its free acidform

The crude purple solid (25 g.) obtained by alkaline n-butanol extractionas described in Example 7 was stirred in 1 liter H₂ O at 25° until itdissolved. The purple solution was adjusted downward in pH from 8.3 to1.1 by dropwise addition of concentrated HCl with stirring. Figaroicacid complex free acid formed as a fine, silt-like brick-red precipitateand was collected by centrifugation. After drying it weighed 13.6 g. andwas active against the L-1210 tumor system in mice down to 0.2mg./kg./day dosage. The supernatant liquor was lyophilized to yield 10.0g. of totally inactive amorphous solids containing 7.8% ash by ignition.The results of the combustion analysis indicate that the cationicmaterial bound to the purple anionic figaroic acid complex is largelyorganic.

EXAMPLE 11 Conversion of Figaroic Acid Complex to Carminomycin I

A suspension of figaroic acid complex (100 g.) in 500 ml. of methanol:chloroform (4:1) was stirred 10 min. and then filtered. The filter cakewas washed three times with 100 ml. portions of methanol. The filtrateand washings were reduced to a volume of 500 ml. at reduced pressure. Tothis solution was added 10 ml. concentrated HCl with stirring. Thesolution was stirred 30 min. at 23° C. and then poured into 3 liters ofchloroform. Distilled water (4 liters) was added to the solution andformation of a precipitate was observed. The resulting suspension wasshaken, filtered and the layers separated. The organic layer was driedover Na₂ SO₄ and evaporated. The aqueous layer was adjusted to pH 7-8with NaHCO₃ and extracted with 5×500 ml. of chloroform. The chloroformextracts were dried over Na₂ SO₄ and evaporated to yield 1.515 g. of asolid which was shown by thin layer chromatography (silica gel plates -*LQD from Quanta/Gram using a developing system of chloroform: methanol:formic acid--85:15:1) to consist primarily of carminomycin I bycomparison with an authentic sample. Similarly, high pressure liquidchromatography indicated the sample to be largely carminomycin I.

EXAMPLE 12 Preparation of Carminomycin I from Figaroic Acid Complex A.Preparation of Partially Purified Figaroic Acid Complex

Figaroic acid complex (400 g.) was stirred with 2500 ml. water for 15min. and then filtered. The procedure was repeated and the filtratesthen combined. The filter cake was stirred with 2500 ml. methanol for 15minutes and filtered. The cake was washed with 2500 ml. methanol and thecombined filtrate evaporated to give 42.7 g. of a solid shown bybioassay to contain from 50-70% of the total bioactivity in the originalfigaroic acid complex sample (hereinafter referred to as solid "A"). Thefilter cake was washed with chloroform until it was colorless, and thecombined filtrate was evaporated to yield 16.4 g. of a solid (solid"B"). Thin layer chromatography of solid "A" indicated the presence of asubstantial amount of ε-rhodomycinone impurity in the sample. Solid "A"was suspended in 100 ml. of chloroform for 10 minutes and filtered. Thisprocedure was repeated six times. Evaporation of the chloroform filtrategave 20 g. of a solid rich in ε- rhodomycinone. The solids from thefiltration step were combined (8 g. total solids) and shown by highpressure liquid chromatography to be partially purified figaroic acidcomplex (low in ε-rhodomycinone impurity).

B. Hydrolysis of Figaroic Acid Complex to Carminomycin I

To 1.0 g. of the partially purified figaroic acid complex prepared inStep A above in 50 ml. of methanol: chloroform (9:1) was added 1 ml. ofconcentrated hydrochloric acid. After letting the solution stand for 90min. at 23° C., crude carminomycin I (0.185 g.) was recovered followingthe procedure of Example 11.

The above hydrolysis procedure in Step B was scaled up using 2 g. offigaroic acid complex and a hydrolysis period of 30 min. The yield ofcarminomycin I was 0.370 g.

EXAMPLE 13 Preparation of Carminomycin I Hydrochloride

To a solution of arminomycin I (prepared according to Example 12) in 10ml. chloroform and 2 ml. methanol was added 2 ml. of 5% methanolichydrochloric acid. The solution was allowed to stand for 1 min. and25ml. diethyl ether then added. The solution was filtered to yield 0.885g. of crude carminomycin I hydrochloride. A 100 mg. sample of the crudesolid was dissolved in 20 ml. water and extracted three times with ethylacetate. The aqueous phase was lyophilized to yield 85mg. ofcarminomycin I hydrochloride.

EXAMPLE 14 Purification of Carminomycin I

A solution of crude carminomycin I (1 g.) in 20 ml. of methanol:chloroform (1:1) was layered onto a column of Sephadex LH-20 (700 g. dryweight swollen with chloroform-column dimensions 5 cm.×170 cm. - bedheight=130 cm.). Chloroform was passed through the column at the rate of8 ml./min. After four hours the flow rate was slowed to 4.6 ml./min. andthe eluant collected using a fraction collector (9.2 ml. fractions takenevery 2 min.). A total of 500 fractions were taken. The cuts were madeas indicated in the table below.

    ______________________________________                                        Sephadex LH-20 Chromatography                                                 of Crude Carminomycin I                                                       Fraction No.    Tubes       Wt.(mg.)                                          ______________________________________                                        1                1-40       175.6                                             2               41-46       21.9                                              3               47-61       84.9                                              4               62-70       12.8                                              5               71-86       30.6                                              6               87-94       6.2                                               7                95-106     16.3                                              8               107-116     16.7                                              9               117-130     17.3                                              10              131-144     40.2                                              11              145-150     50.4                                              12              151-158     36.5                                              13              159-171     194.4                                             14              172-180     25.7                                              15              181-200     38.1                                              16              201-240     36.1                                              17              241-324     43.0                                              18              325-420     60.7                                              19              420-500     26.4                                              ______________________________________                                    

Fraction No. 13 proved to be pure carminomycin I by use of NMRchromatography and biassay (ILB, BS-8 and L1210).

We claim:
 1. A biologically pure culture of the microorganismStreptosporangium sp. ATCC 31129, said culture being capable ofproducing the antibiotic complex, figaroic acid complex, in arecoverable quantity upon cultivation in an aqueous nutrient mediumcontaining assimilable sources of nitrogen and carbon.